1. Field of the Invention
The present invention relates to the field of synthesis of peptides and related compounds, using solid supports, and cleavage of such compounds through cleavage of a diazo bond, releasing at least a portion of the compound from the support.
2. Related Art
One of the primary goals for the field of proteomics is finding ways to enrich specific protein targets from complex mixtures. Generally, this is accomplished with small molecular tags that allow specific modification by the formation of a stable covalent bond with reactive groups on the target protein.1 The proteomic probes can either be generally reactive towards free nucleophiles such as thiols (i.e., ICAT reagents)2 or react through a specific enzymatic process with a key catalytic residue.3 The labeled targets can then be enriched using a affinity purification methods, which mainly exploit the diffusion limited binding of biotin to (immobilized) streptavidin. Although this method allows efficient isolation of even highly dilute targets, one of the primary limitations is the need for harsh, denaturing conditions to disrupt the biotin-streptavidin interaction. The elution conditions generally result in contamination of the desired probe labeled proteins with avidin monomers, proteins that were non-selectively bound to the streptavidin, and endogenously biotinylated proteins. Therefore, the incorporation of a cleavable linker between the biotin tag and the site of attachment to the target protease will be of great value, since it allows specific elution of proteins or peptides that were labeled by a given probe. Furthermore, specific cleavage of the probe structure can be used to reduce the size of the chemical modification on the target protein leading to enhanced mass spectrometry characteristics.
Recently, a number of cleavable linkers have been reported, with a focus on applications in mass spectrometry and ICAT.4-6 ICAT reagents consist of conjugate molecules containing an affinity tag (e.g., biotin), an isotope tag and a protein-reactive group such as an iodoacetamide for attachment to cysteine SH groups in proteins. A non-deuterated and deuterated pair of ICAT reagents are used to determine the relative levels of proteins in complex protein mixtures. However, these regents require strong acid (TFA), making cleavage of labeled proteins directly from strepavidin resin problematic.
Other Patents and Publications
US 2002/0076739 to Aebersold, et al., published Jun. 20, 2002, entitled “Rapid quantitative analysis of proteins or protein function in complex mixtures,” relates to the above-mentioned ICAT method. It discloses analytical reagents and mass spectrometry-based methods that employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). Thus, the linker can be cleavable, for example, by chemical, thermal or photochemical reaction. Photocleavable groups in the linker may include the 1-(2-nitrophenyl)-ethyl group. Thermally labile linkers may, for example, be a double-stranded duplex formed from two complementary strands of nucleic acid, a strand of a nucleic acid with a complementary strand of a peptide nucleic acid, or two complementary peptide nucleic acid strands which will dissociate upon heating. Cleavable linkers also include those having disulfide bonds, acid or base labile groups, including among others, diarylmethyl or trimethylarylmethyl groups, silyl ethers, carbamates, oxyesters, thiesters, thionoesters, and α-fluorinated amides and esters.
US 2006/0147985 to Barone, et al., published Jul. 6, 2006, entitled “Methods and compositions for monitoring polymer array synthesis,” discloses cleavable linkers useful in monitoring polymer synthesis in an array on a substrate, exemplified by the photocleavable group MeNPOC.
US 2005/0010059 to Beauchamp, et al., Jan. 13, 2005, entitled “Chemical reagents capable of selective attachment to and reaction with peptides and proteins,” discloses crown ethers capable of selectively forming non-covalent complexes and initiating intermolecular reactions with amine-containing compounds. The disclosure includes a method of covalently attaching amino acids via carbene insertion chemistry comprising adding a compound to the amino acid, where the compound comprises at least one crown ether group and a diazo group.
U.S. Pat. No. 6,951,682 to Zebala, issued Oct. 4, 2005, entitled “Porous coatings bearing ligand arrays and use thereof,” discloses photoresist compounds of the formula >C═N2 in which the compounds are converted by light to >COOH+N2. These compounds have a ketone group adjacent to the diazo group. As stated there, the photolytic response of phenolic photoresists reflects the photochemistry of the photosensitive diazoquinone often also referred to as a diazoketone, diazo-oxide, diazoanhydride, or quinone diazide.
U.S. Pat. No. 6,818,420 to Chou, et al, issued Nov. 16, 2004, entitled “Methods of using FET labeled oligonucleotides that include a 3′-5′ exonuclease resistant quencher domain and compositions for practicing the same,” discloses a dark quencher having a formula wherein one or more substituted aryl groups comprise the linkage —N═N-aryl. The substituted aryl has substituents R2 through R5 which may be independently —H, halogen, —O(CH2)nCH3, —(CH2)nCH3, —NO2, SO3, —N[(CH2)nCH3]2 wherein n=0 to 5 or —CN.
U.S. Pat. No. 4,279,998 to Shahani, et al., issued Jul. 21, 1981, entitled “Regenerable insoluble support for protein immobilization,” discloses an insoluble support for immobilized proteins in which the protein is connected to the support by a spacer arm containing a diazo linkage, and, when the protein is denatured, the spacer arm is broken by reducing the diazo linkage to remove the denatured protein. Activated p-(N-acetyl-L-tyrosine azo) benzamidoethyl agarose beads were prepared, comprising a linkage of -benzyl-N═N=2-hydroxy, 5-heteralkly substituted benzyl.
Kemp and Scott, “Ehrlich chromogens, probable cross-links in elastin and collagen,” Biochem. J. (1988) 252, 387-393 discloses an affinity support method, utilizing a polyacrylamide substrate to which a diazobenzene group is linked via an alkali-labile phenol ester.
Denny and Blobel, “125I labeled crosslinking reagent that is hydrophilic, potoactivatable, and cleavable through an azo linker,” Proc. Nat. Acad. Sci. 81:5286-5294 (1984) discloses a radioactive cross linker, N-[4-(Pazido-m-[1251]iodophenylazo)benzoyl]-3-aminopropyl-N′-oxysulfosuccinimide ester, which contains an aryl-N═N-aryl structure. As a model system, the derivatized protein A-Sepharose was added to human serum to determine if radioactive label could be specifically transferred from protein A to the heavy chain of IgG following photocrosslinking and cleavage of the crosslinks with sodium dithionite.